GST-Tag antibody

Antigen: GST-Tag

Clonality: Monoclonal (3B2)

Host: Mouse

Application: Western Blotting (WB), Immunoprecipitation (IP)

Catalog no.: ABIN131909

Additional Information

Immunogen: This antibody was purified from mouse ascites fluid using Protein A sepharose. This hybridoma was established by fusion of mouse myeloma cell SP2/0-Ag 14 with BALB/c mouse splenocyte immunized with recombinant GST protein.

Format: Liquid

Isotype: IgG2b  (Matching secondary antibodies)

Clone: 3B2

Description: Expression vectors containing a protein and a tag protein are commonly used. GST-Tag fusion protein expression system is preferably used in various laboratories, because its simple protein purification step by an affinity chromatograpy. This specific antibody for GST -Tag fusion protein is useful tools for monitoring of the fusion protein expression and affinity purification.

Specificity: This antibody recognizes recombinant GS T-Tag specifically.

Application Details

Application Notes: SDS PAGE & Western Blotting 1) Boil the sample for 3~5 minutes and centrifuge at 12,000 x g for a minute. Other methods may be employable. 2) Resolve 10 µ l of sample by SDS -polyacrylamide gel electrophoresis (See the manufacture's manual for electrophoresis condition). 3) Transfer to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system. (Transfer Buffer - 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure. 4) The transferred proteins can be visualized by staining the membrane for 1 minute with Ponceau (SIGMA, Cat# P-7170). Rinse the membrane with PBS. 5) Non-specific binding sites are blocked by immersing the membrane in 5% Skim Milk/PBS/0.05% Tween 20 for 1 hr.at room temperature (20-25 o C) or for overnight at 4 o C. 6) Incubate in primary antibody diluted as suggested in the Applications for 1 hour at room temperature(20 - 25 o C). (The concentration of the antibody to be used will be dependent on condition.) 7) Wash the membrane 3 times with PBS containing 0.05% Tween20 for 5~10 minutes each. 8) Incubate the membrane with secondary antibody (1:10,000 diluted horseradish peroxidase conjugated anti mouse IgG (H+L) antibody in PBS containing 0.05% Tween 20) for 45 minutes at room temperature (20-25 o C). 9) Wash the membrane 3 times with PBS containing 0.05% Tween20 for 5~10 minutes each. 10) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 11) Expose to a X-ray film in a dark room for 30 seconds. Develop the film as usual. The condition for exposure and development may vary. Immunoprecipitation 1) Add 5 µ g of the antibody to 100 µ l of E. Coli extracts containing GST -Tag fusion protein. Mix well and incubate with gentle agitaiton for 30- 120 minutes at 4 o C. 2) Add 20 µ l of 50 % Protein A-agarose beads. Mix well and incubate with gentle agitation for 1 hour at 4 o C. 3) Wash the beads 4 times with ice-cold Lysis buffer (50 mM Hepes pH 7.3, 250 mM NaCl, 0.2% NP- 40, 5 mM EDTA, 10% glycerol), then centrifuge the tube at 2,500 x g for 10 seconds to remove the buffer. 4) Resuspend the beads in 40 µ l of Laemmli SDS PAGE sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Apply 20 µ l/lane for the SDS -PAGE analysis (See SDS-PAGE & Western blotting. ) DATA:Western blotting 1/2 Lane 1: Anti-GST-Tag Recombinant GST 94 67 43 30 20 14.4 Western blotting: 1 µ g/ml. Immunoprecipitation: 5 µ g/100 µ l of E. Coli extracts containing GST -Tag fusion protein. Detailed procedures are provided in the following Research Applications.

Buffer: 100 micrograms of IgG in 100 microliter volume of PBS containing 50% glycerol. No preservative is contained.

Restrictions: For Research Use only

Images

Western Blot